Project Summary The objective of this proposal is to develop new tools to independently and spatiotemporally manipulate distinct epigenetic regulatory mechanisms at defined gene loci. Epigenetic modifications and chromatin structures regulate gene activity. Alteration of epigenetic pathways and mutations in epigenetic regulators contribute to many developmental diseases and cancers. DNA methylation and histone modification have been shown to play important roles in gene activation. Several studies suggest that these epigenetic pathways are orchestrated to regulate activity-gene expression. However, current methods are limited to precisely dissect the functional relationship between these distinct epigenetic regulatory mechanisms. To address this limitation, we propose to develop unique chemical and light inducible methods that integrate dCas9/gRNA-guided targeting with the chemically induced proximity (CIP) technology to individually or simultaneously control different layers of epigenetic regulations (e.g., H3K27 acetylation and DNA demethylation). We will use this new technology to study their functional interplays and test multiplex epigenetic editing in the epigenome. After finishing this work, we expect to establish unique tools that will significantly contribute to the studies of epigenetic regulation in gene activation and offer new directions in developing new epigenetic-based therapies.